A subculture of various cells such as adherent cells and suspended cells has been conventionally conducted. For example, cells which are adhering to a culturing container and proliferated, are released therefrom by a physical method using a cell scraper or a pipette, or a physiological method using an enzyme, and then the cells are seeded in a new culturing container having a fresh culture medium and conducted with subculture. Especially, because pluripotent stem cells which are the adherent cells easily result in cell death when purifying into single cells, it is necessary to conduct the subculture while maintaining cell masses. For instance, the pluripotent stem cells are released as colonies, and the cell masses having appropriate size are disrupted by using a pipette etc., and then seeding it into a new culturing container to conduct the subculture thereof.
In the subculture, when a releasing means as the physical method is used, problems that the cells are excessively damaged are occurred. When the other releasing as the physiological method is used, the cells are damaged. Although this damage is smaller than that occurring from the physical method, the physiological method causes problems in which an enzyme reaction with respect to the cells is non-uniform and the cells are dead after differentiation into the single cells. Further, when the pipetting procedure is conducted, the size of the cell masses is varied and the cell masses having a uniform size is difficulty obtained. Thereby size of the colonies which is produced after the subculture is dispersed. According to the problems of the dispersion of the size of the respective colonies, even when the some colonies reach a specific size, an insufficient culture state of the culture of others is occurred. The dispersion of the size thereof adversely affects quality control of the cells and causes decrease of productive efficiency.
A method improving damage of the cells by the releasing means and non-uniformity of the colonies in the subculture is disclosed in Patent Document 1. Patent Document 1 discloses a substrate for holding or amplifying and culturing pluripotent stem cells, which includes a nanofiber consisting of a biopolymer, and a culturing method using the same. According to using the substrate, the pluripotent stem cells may be dispersed to the single cells by slight handling by the pipetting procedure without an enzyme treatment when conducting the subculture. Uniformed cells may be obtained while decreasing a cell death rate.
In addition, the pluripotent stem cells are easily differentiated. The pluripotent stem cells, which initiate differentiation, difficultly restore to a state of undifferentiation. A maintenance culture therefore needs to be conducted without changing to state which easily differentiates the cells. Further, when the differentiation of the cells is initiated while culturing it, it is necessary to remove the cells which initiate the differentiation from the culturing container because the other cells of circumference thereof are adversely affected, and a yield and purity of the undifferentiated cells are decreased.
In order to culture the cells, an opened-system culture and a closed-system culture have been used. According to the conventional opened-system culture using dishes, work of medium replacement of culture is carried out while opening a cover of the dishes. Therefore the cells which initiate the differentiation may be aspirated and removed by inserting an aspirator or a micropipette thereinto. The opened-system culture is inexpensive and has excellent operability and thus, it is useful for research.
On the other hand, according to the closed-system culture, because the aspirator cannot be inserted thereinto, it is difficult to selectively remove the cells which initiate the differentiation. In contrast, according to the closed-system culture, an incidence of contamination and infection risk may decrease than that of the opened-system culture. Therefore the closed-system culture is useful for medical care. Technology and structure of the closed-system culture, which is capable of selectively removing the cells initiating the differentiation by simple work, have been demanded.
As a method which is capable of selectively removing and recovering the cells in the closed-system culture, for example, a method for regionally and selectively releasing the cells from an adhesive face by high frequency vibration is disclosed in Patent Document 2. According to the method, the cells may be selectively released from an adhering face of the culturing container of the closed-system or the opened-system through a non-contact means.
Regardless of the closed-system culture and the opened-system culture, according to a cell culture method and a subculture using a container such as a multi plate of which wells as concave having a dented concave part for forming the cell clusters are molded therein, the container having excellent visibility is required in order to observe the cells in the wells. The cell culture method comprises introducing cell suspension which disperses the cells for culturing into the container; seeding the cells which are precipitated in the wells; forming the cell clusters and culturing them; and recovering the cultured cells by the handling using the pipette, by the high frequency vibration or by sending a cell-releasing solution as needed. The subculture may be carried out by repeating these steps.
The shape fixed container in which the wells are preliminarily molded has a problem of difficult observation, because visibility is insufficient due to a concave-convex shape of the container. When the cell suspension is introduced into the container, air bubble is easily mixed thereinto. The visibility therefore is further decreased, because an establishing position of the cell becomes an unstable state by existence both of the cells and the air bubble in the wells, and air bubble varies transmittance and a refractive index of light.
As described above, the various subcultures have been conventionally conducted. It have been demanded that a container and cell culture method using the same, which can solve the above problems, can culture the pluripotent stem cells of huge amount while maintaining the undifferentiated state thereof, can conduct observation and examination of a state of the cells from the outside direction of the container, and can perform a homogeneous subculture with more excellent efficiency.